Efficient and regulated import of long chain fatty acids (LCFAs) into specific tissues is critical for normal lipid homeostasis. Mismatch between LCFA import and utilization in the pancreas, skeletal muscle, vascular endothelial cells, and the heart may play an important role in the pathogenesis of diabetes and heart failure. When present at high, pathophysiologic concentrations, un-ionized LCFAs undergo rapid translocation from one leaflet of the bilayer to the other by a non- protein-mediated mechanism. However, evidence is emerging that proteins play an important role in the trafficking of LCFAs across the plasma membrane of cells at low, physiologic LCFA concentrations. In previous work, we identified the long-chain fatty acid transport protein (FATP1) and acyl-CoA synthetase (ACS1) as proteins that facilitate LCFA transport into cells. This project is designed to molecularly characterize FATP1 and its role in vectorial fatty acid transport across the membranes of mammalian cells. We will test the hypothesis that FATP1 functions as a component of an oligomeric complex that transports and esterifies LCFAs. In Specific Aim 1 we will analyze structure-function correlates for FATP1. In Specific Aim 2 we will characterize the FATP1 oligomeric complex. In Specific Aim 3 we will evaluate the role of esterification in FATP1-mediated transport. Together these studies will help to define the molecular mechanisms by which LCFAs are transported across the plasma membrane of mammalian cells. Characterization of these mechanisms has relevance to common human diseases such as diabetes and heart failure in which abnormal lipid homeostasis contributes to pathogenesis.